Streptococcus pneumoniae is frequently isolated from the young, the elderly, and the immunocompromised as the etiologic agent of a broad range of diseases (Centers for Disease Control and Prevention, “Prevention of pneumococcal disease: Recommendations of the Advisory Committee on Immunization Practices” (ACIP), 1997 Morbid. Mortal. Weekly Rep. (RR-8) 46:1-24). A number of diagnostic assays for the detection of Streptococcus pneumoniae have been developed and are described in the literature. Unfortunately they are not sufficiently definitive, reliable, or sensitive to be used on a routine basis (Sampson et al. 1994, “Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins”, Infect. Immun. 62:319-324). The large number of pneumococci currently known (90 different serotypes) increases the challenge of diagnosis and further complicates assay development and vaccine development.
Streptococcus pneumoniae normally colonizes the nasopharynx and can be found in 5-10% of healthy adults and 20-40% of healthy children. S. pneumoniae is responsible for a variety of infections including common “strep throat,” otitis media, sinusitis, meningitis, tracheitis, bronchitis, rheumatic fever, and community-acquired pneumonia. It can also spread from the nasopharyngeal cavity into the central nervous system, heart valves, bones, joints, and peritoneal cavity. The organism typically binds to the upper respiratory tract. It is transmitted person-to-person via droplets in the air; transmission through direct contact is also thought to occur.
Generally streptococcal infections affect the lungs in less than 5% of cases. Such lung infections, however, progress with great rapidity and severity, despite treatment with antibiotics. This form of pneumonia is difficult to control and may last for weeks. Although severe pneumonia can be caused by a wide variety of bacteria, as well as by viruses, streptococcal infection accounts for about 55% to 76% of cases. About 2 million Americans get pneumonia each year, with 40,000 to 70,000 deaths reported. Pneumonia ranks sixth among all categories of disease as a cause of death. Despite the many testing technologies available to physicians, it is sometimes very difficult to determine which species of bacteria is responsible for the illness; in up to half the cases no etiological agent can be identified.
Considerable effort has been invested in developing a vaccine against Streptococcus pneumoniae. An immunogenic species-common protein has been identified (Russell et al. 1990, “Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae”, J. Clin. Microbiol. 28:2192-2195; U.S. Pat. No. 5,422,427). In these references, the 37 kDa protein of the present invention is referred to pneumococcal fimbrial protein A. This 37 kDa S. pneumoniae protein has been the focus of several studies and is now designated pneumococcal surface adhesin protein A (PsaA). Immunoblot analysis studies using anti-PsaA monoclonal antibody showed that PsaA is common to all 23 pneumococcal vaccine serotypes known at that time (i.e., about 1990). Enzyme-linked-immunosorbent assay studies have indicated that patients with pneumococcal disease show an antibody increase in convalescent-phase serum to PsaA compared with acute-phase serum antibody levels (Tharpe et al. 1995, “Purification and seroreactivity of pneumococcal surface adhesin A (PsaA)”. Clin. Diagn. Lab. Immunol. 3:227-229; Tharpe et al. 1994, “The utility of a recombinant protein in an enzyme immunoassay for antibodies against Streptococcus pneumoniae”, Abstr. V-2, p. 617,1994. American Society for Microbiology, Washington, D.C.). Additionally a limited in vivo protection study showed that antibodies to the 37-kDa protein protect mice from lethal challenge (Talkington et al. 1996, “Protection of mice against fatal pneumococcal challenge by immunization with pneumococcal surface adhesin A (PsaA)”, Microbial Pathogenesis 21:17-22).
The gene encoding PsaA from S. pneumoniae strain R36A (an unencapsulated strain) has been cloned in E. coli and sequenced. This serotype does not, however, contain a nucleic acid encoding a 37 kDa protein that is highly conserved among the various serotypes (Sampson et al. 1994). This particular nucleic acid and polypeptide, therefore, are of limited value for use as diagnostic reagents, in prevention or treatment of infection, or in vaccine development.
U.S. Pat. No. 5,854,416 provides a nucleic acid encoding the 37-kDa protein from serotype 6B of Streptococcus pneumoniae. Isolated nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein are also provided. The patent further discloses purified polypeptides encoded by the nucleic acid encoding the 37-kDa protein, as well as nucleic acids comprising a unique fragment of at least 10 nucleotides coding for the 37-kDa protein. In addition, antibodies are provided which selectively bind the polypeptides encoded by the nucleic acid encoding the 37-kDa protein and the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37-kDa protein. The patent additionally provides vaccines comprising immunogenic polypeptides encoded by the nucleic acid encoding the 37-kDa protein and by the nucleic acids comprising a unique fragment of at least 10 nucleotides of the 37 kDa protein. Further provided is a method of detecting the presence of Streptococcus pneumoniae in a sample comprising the steps of contacting a sample suspected of containing Streptococcus pneumoniae with nucleic acid primers capable of hybridizing to a nucleic acid comprising a portion of the nucleic acid encoding the 37-kDa protein, amplifying the nucleic acid and detecting the presence of an amplification product, the presence of the amplification product indicating the presence of Streptococcus pneumoniae in the sample. Further provided are and methods of detecting the presence of Streptococcus pneumoniae in a sample using antibodies or antigens, methods of preventing and treating Streptococcus pneumoniae infection in a subject.
Sampson et al. (1997, “Limited diversity of Streptococcus pneumoniae psaA among pneumoccocal vaccine serotypes”. Infection and Immunity, 65(5):1967-1971) show that the pneumococcal surface adhesin A (PsaA) protein belongs to a group of proteins designated the lipoprotein receptor 1 antigen family. The gene encoding PsaA from an encapsulated strain of pneumococcal serotype 6B was cloned and sequenced. The peptide sequence was compared to that of homologs found in S. pneumoniae serotype 2, viridans streptococci, and Enterococcus faecalis. Identity values among the deduced peptides ranged from 57 to 98%. The polymorphism of psaA was examined among the 23 encapsulated vaccine serotypes by using PCR-restriction fragment length polymorphism analysis. This analysis showed that restriction sites within the gene were highly conserved. The lack of variation for the other restriction sites within the gene indicates that PsaA is genetically conserved, an important characteristic necessary for a candidate common protein vaccine.
A pneumnococcal polysaccharide vaccine was developed with 23 types of pneumococcal bacteria which cause 88 percent of bacteremic pneumococcal disease. Vaccinated, healthy adults are protected from some or all pneumococcal pneumonia bacteria types. However, the carriage rates of the organism do not decline significantly in vaccinated people. Additionally, adverse reactions from this vaccine may occur in older persons, children under the age of two years, and those stricken with long-term illnesses. Furthermore, it is found that the polysaccharide vaccine fails to provide protection in young children, the elderly and the immunocompromised. This led to development of a second-generation protein-conjugate vaccine. This vaccine, composed of the seven most frequent invasive disease-causing capsular serotypes, may overcome the problems of poor immunogenicity associated with the 23-valent vaccine. However, there are indications that this protein-conjugate vaccine may not prevent replacement carriage of serotypes not contained in the vaccine (Mbelle et al. 1997. “Immunogenicity and impact on carriage of 9-valent pneumococcal conjugate vaccine given to infants in Soweto, South Africa”, Abstr. L8-12, p. 13, In Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Washington, D.C.; Obaro et al. 1996. “Carriage of pneumococci after pneumococcal vaccination”, Lancet 348:271-272). These concerns, along with reports of an increase in antibiotic-resistant pneumococci (Centers for Disease Control and Prevention, 1997), have shifted interest toward the development of a vaccine based on immunogenic pneumococcal species-common proteins of S. pneumoniae. The most promising of these proteins include pneumococcal surface adhesin A (Russell et al. 1990, “Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae”, J. Clin. Microbiol. 28:2192-2195).
PsaA is under study both as vaccine immunogen and as a reagent for diagnostic assay development (Tharpe et al. 1998, “Comparison of a pneumococcal common protein (PsaA) antibody ELISA and a PsaA immune complex ELISA for detection of pneumococcal serum antibody”, Pathobiology, 66:77-83). Monoclonal antibody studies suggest that PsaA is expressed on all 90 serotypes of S. pneumoniae (Crook et al. 1998, “Immunoreactivity of five monoclonal antibodies against the 37-kilodalton common cell wall protein (PsaA) of Streptococcus pneumoniae”, Clin. Diagn. Lab. Immunol. 5:205-210) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the 23 vaccine serotypes demonstrated the conservation of the psaA gene (Russell et al. 1990).
A culture assay is currently the Gold Standard for detecting Streptococcus pneumoniae. Patient sputum, throat swab, or blood specimens can be cultured for Streptococcus pneumoniae if they are handled correctly. Such diagnostic tests, however, are generally time-consuming, requiring at least overnight incubation. Furthermore, cultures also lack the certainty of positive identification, providing positive diagnoses only 20% to 30% of the time. Thus the current Gold Standard diagnostic assay has important deficiencies associated with it. Because of the speed with which streptococcal pneumoniae can develop and potentially lead to death, there is a need for rapid testing resulting in earlier treatment of infection. This has the potential to significantly abbreviate the course of the disease and reduce mortality.
Rapid assays for Streptococcus are available in a number of formats. Older format kits involve the use of a multistep latex agglutination procedure or an ElA format, and employ samples such as serum, whole blood, or sputum swabs. Most take less than eight minutes to perform, and show results through the appearance of a colored visual indicator.
In order to detect all 90 serotypes of S. pneumoniae using currently employed procedures, assay reagents representing the 90 different components, such as are used in serologic assays including latex agglutination and counterimmunoelectrophoresis, would be needed. Pneumococcal diagnostic assays are based primarily on detection of pneumococci, pneumococcal antigens, DNA or RNA in blood or body fluids. Although blood culture is currently the most accurate method by which to diagnose pneumococcal disease, only approximately 30% of specimens test positive with this method (Kalin et al. 1983, “Diagnosis of pneumococcal pneumonia: a comparison between microscopic examination of expectorate, antigen detection, and cultural procedures”, Scand. J. Infect. Dis. 15:247-255). Conventional serologic methods lack uniform diagnostic sensitivity and specificity and are time-consuming because they require reagents in which all serotypes are represented. Although PCR assays have been reported in the literature (Toikka et al. 1998, “Pneumolysin PCR-based diagnosis of invasive pneumococccal infection in children”, J. Clin. Microbiol. 37:633-637), they have not been widely adapted for use in the clinical laboratory because they have not been shown to be consistently and uniformly sensitive. Toikka et al. (1998), for example, described the necessity of testing three different blood fractions to maximize sensitivity. They also suggested that a combination of other diagnostic methods is needed for diagnosis of invasive pneumococcal disease.
Automated serological testing is used in diagnostic testing for Streptococcus pneumoniae infection. These assays identify and quantify the existence of antibodies to bacterial pathogens. All antibody detecting assays may miss active infection if the level of antibodies in a patient's blood is too low, as occurs with certain immunocompromised individuals, or if infection is too recent for antibodies to have been produced. Additionally, as with all antibody-based assays, because levels of antibodies produced against a previous infection fall slowly, testing may indicate an infection that no longer exists.
Thus, there remains a need for an universal, highly sensitive, and highly species-specific method of detection that will permit straightforward and reliable diagnosis of pneumococcal disease. There is further a need for specific reagents that enable universal and specific assays for Streptococcus pneumoniae to be carried out. There further remains a need for polypeptides that are immunogenic for stimulating an immune response against Streptococcus pneumoniae. Additionally there remains a need for a nucleic acid that includes a sequence encoding such an immunostimulatory polypeptide. The present invention recognizes these needs, and provides compositions and methods based on psaA genes and PsaA polypeptides to address these needs.